Rational design of a calcium-binding protein

Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The resulting protein selectively binds calcium over magnesium with calcium-binding affinity comparable to that of natural extracellular calcium-binding proteins (K(d) of 50 microM). This experiment is the first successful metalloprotein design that has a high coordination number (seven) metal-binding site constructed into a beta-sheet protein. Our results demonstrate the feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators. The resulting site exhibits strong metal selectivity, suggesting that it should now be feasible to understand and manipulate signaling processes by designing novel calcium-modulated proteins with specifically desired functions and to affect their stability.     

De novo design of a redox-active minimal rubredoxin mimic

Metal-binding sites in metalloproteins frequently occur at the interfaces of elements of secondary structure, which has enabled the retrostructural analysis of natural proteins and the de novo design of helical bundles that bind metal ion cofactors. However, the design of metalloproteins containing beta-structure is less well developed, despite the frequent occurrence of beta-conformations in natural metalloproteins. Here, we describe the design and construction of a beta-protein, RM1, that forms a stable, redox-active 4-Cys thiolate Fe(II/III) site analogous to the active site of rubredoxin. The protein folds into a beta-structure in the presence and absence of metal ions and binds Fe(II/III) to form a redox-active site that is stable to repeated cycles of oxidation and reduction, even in an aerobic environment.     

Rational design of a macrocycle-based chemosensor for anions

A macrocycle-based fluorescence chemosensor has been designed and synthesized from the reaction of dansyl chloride and a hexaaminomacrocycle containing four secondary and two tertiary amines. The new chemosensor has been examined for its binding ability towards phosphate, sulfate, nitrate, iodide, bromide, chloride, and fluoride by fluorescence spectroscopy in DMSO. The results indicate that the compound binds each of the anions with a 1:1 stoichiometry, showing high affinity for oxoanions, chloride, and iodide with binding constants up to four orders of magnitude. Ab initio calculations based on density functional theory (DFT) suggest that the ligand is deformed in order to encapsulate an anion, and each anion, except fluoride, is bonded to the macrocycle through two NH?X⁻ and four CH?X⁻ interactions.     

Novel design of a pentacyclic scaffold as structural mimic of saframycin A

The design, synthesis and evaluation of a pentacyclic scaffold, CWO-324 to mimic saframycin A is described. CWO-324 is readily synthesized in five steps from 1,4-diacetyl-piperazine-2,5-dione and 2,5-dimethoxybenzaldehyde. CWO-324 was found to scission DNA, binds to bases 69-83(5′-GCAGTCAGG CACCGT-3′) of Hind III/Rsa I from plasmid pBR322 DNA in a foot-printing study and possesses anti-tumor activity.     

Rational design and synthesis of a novel class of active site-targeted HIV protease inhibitors containing a hydroxymethylcarbonyl isostere. Use of phenylnorstatine or allophenylnorstatine as a transition-state mimic.

A novel class of HIV-1 protease inhibitors containing a hydroxymethylcarbonyl (HMC) isostere were designed from the substrate transition state and synthesized. Phenylnorstatine [Pns; (2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] and the 2S diastereomer, (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) were effective transition-state mimics, and incorporation of Pns-Pro or Apns-Pro at the P1-P1' site gave potent and specific HIV-1 protease inhibitors. In the inhibitory assays, the chemically synthesized [Ala67,95] HIV-1 protease was used.     

Rational design and efficient synthesis of a fluorescent-labeled jasmonate

A fluorescent-labeled jasmonate was rationally designed based on examination of the model of interaction between the jasmonate and its receptor. An efficient synthetic route has been developed for this molecule. The biological activity of this fluorescent probe was retained which was similar to that of the methyl jasmonate as examined by root growth inhibition bioassay. This fluorescent probe will greatly facilitate biological studies of jasmonates through fluorescent imaging.     

Rational design of a cyclin A fluorescent peptide sensor

We report the design and development of a fluorescent sensor specifically designed to target cyclin A, a protein that plays a key role in the regulation of the cell cycle. Computational studies provide a molecular picture that explains the observed emission increase, suggesting that the 4-DMAP fluorophore in the peptide is protected from the bulk solvent when inserted into the hydrophobic binding groove of cyclin A.     

Rational structure-based design of a novel carboxypeptidase R inhibitor

A novel carboxypeptidase R (CPR) inhibitor, related to potato carboxypeptidase inhibitor (PCI), was designed using rational structure-based strategies, incorporating two principle facts: CPR has a strong affinity for basic amino acids, and the two lysine and arginine residues of PCI are orientated in the same direction and held in close spatial proximity by three disulfide bonds. Initially, a disulfide-bonded fragment of PCI was synthesized showing weak competitive inhibitory activity against CPR. Subsequently, a smaller linear 9-mer peptide, designated CPI-2KR, was designed/synthesized and found to be a more efficient competitive inhibitor of CPR, without affecting the activity of the other plasma carboxypeptidase, carboxypeptidase N. In vitro studies showed that, together with tissue plasminogen activator, CPI-2KR synergistically accelerated fibrinolysis, representing a lead compound for the design of smaller organic molecules for use in thrombolytic therapy.     

Rational design of porous titanophosphates

To rationally design new nanoporous materials based on titanophosphates, a small library of titanium phosphate crystalline nanoporous compounds has been build up and its compounds have been investigated by X-ray diffraction and by in- and ex-situ NMR. The main trends of the unusual titanium solution chemistry, of the prenucleation building units and of their assembling have been established. The classical trial and error strategy can therefore be replaced by a better control of the steps leading to the final targeted network.     

Rational design of DNA nanoarchitectures

DNA has many physical and chemical properties that make it a powerful material for molecular constructions at the nanometer length scale. In particular, its ability to form duplexes and other secondary structures through predictable nucleotide-sequence-directed hybridization allows for the design of programmable structural motifs which can self-assemble to form large supramolecular arrays, scaffolds, and even mechanical and logical nanodevices. Despite the large variety of structural motifs used as building blocks in the programmed assembly of supramolecular DNA nanoarchitectures, the various modules share underlying principles in terms of the design of their hierarchical configuration and the implemented nucleotide sequences. This Review is intended to provide an overview of this fascinating and rapidly growing field of research from the structural design point of view.     

Rational design of polymer colloids

Recent advances in understanding of the fundamental mechanistic events in emulsion polymerization give the potential for rational design of new materials based on polymer colloids. It is now possible to design a new industrial process from first principles, based on well‐understood mechanistic principles. An overview of recent developments in the fundamental science of emulsion polymerization is given, with examples of the application of this knowledge to topologically‐controlled synthesis of novel materials based on natural rubber and polybutadiene seed latexes.     

Rational design of allosteric ribozymes

Background: Efficient operation of cellular processes relies on the strict control that each cell exerts over its metabolic pathways. Some protein enzymes are subject to allosteric regulation, in which binding sites located apart from the enzyme's active site can specifically recognize effector molecules and alter the catalytic rate of the enzyme via conformational changes. Although RNA also performs chemical reactions, no ribozymes are known to operate as true allosteric enzymes in biological systems. It has recently been established that small-molecule receptors can readily be made of RNA, as demonstrated by the in vitro selection of various RNA aptamers that can specifically bind corresponding ligand molecules. We set out to examine whether the catalytic activity of an existing ribozyme could be brought under the control of an effector molecule by designing conjoined aptamer-ribozyme complexes.Results: By joining an ATP-binding RNA to a self-cleaving ribozyme, we have created the first example of an allosteric ribozyme that has a catalytic rate that can be controlled by ATP. A 180-fold reduction in rate is observed upon addition of either adenosine or ATP, but no inhibition is detected in the presence of dATP or other nucleoside triphosphates. Mutations in the aptamer domain that are expected to eliminate ATP binding or that increase the distance between aptamer and ribozyme domains result in a loss of ATP-specific allosteric control. Using a similar design approach, allosteric hammerhead ribozymes that are activated in the presence of ATP were created and another ribozyme that can be controlled by theophylline was created.Conclusions: The catalytic features of these conjoined aptamer-ribozyme constructs demonstrate that catalytic RNAs can also be subject to allosteric regulation — a key feature of certain protein enzymes. Moreover, by using simple rational design strategies, it is now possible to engineer new catalytic polynucleotides which have rates that can be tightly and specifically controlled by small effector molecules.     

Rational design of a phthalocyanine-perylenediimide dyad with a long-lived charge-separated state

A new ZnPc-PDI dyad presenting for the first time a charge-separated state lower in energy than the triplet excited state of the ZnPc and PDI has been synthesized. The rational design implies the substitution of the ZnPc with phenoxy groups and the bay substitution of the PDI with sulfonyl substituents. The lifetime of the charge-separated state was 72 μs.     

Rational design of a reversible pH-responsive switch for peptide self-assembly

Peptide TZ1H, based on the heptad sequence of a coiled-coil trimer, undergoes fully reversible, pH-dependent self-assembly into long-aspect-ratio helical fibers. Substitution of isoleucine residues with histidine at the core d-positions of alternate heptads introduces a mechanism by which self-assembly is coupled to the protonation state of the imidazole side chain. Circular dichroism spectroscopy, transmission electron microscopy, and microrheology techniques revealed that the self-assembly of TZ1H coincides with a distinct coil-helix conformational transition that occurs within a narrow pH range near the pKa of the imidazole side chains of the core histidine residues.     

Rational design of a BiFeWO6 nanostructure for supercapacitor applications

Journal of Solid State Electrochemistry - Scientists are increasingly interested in improving electroactive technologies for supercapacitor applications, since energy storage devices have improved...     

Rational design of a promising oxychalcogenide infrared nonlinear optical crystal

Oxychalcogenides with the performance-advantages of both chalcogenides and oxides are emerging materials class for infrared (IR) nonlinear optical (NLO) crystals that can expand the wavelength of solid-state lasers to IR regions and are of importance in industrial and civil applications. But rationally designing a high-performance oxychalcogenide NLO crystal remains a great challenge. Herein, we chose the melilite-type Sr₂ZnSi₂O₇ as the structure template. Through part isovalent substitution of S²⁻ for O²⁻ anions, the first hetero-anionic thiostannate Sr₂ZnSn₂OS₆ with wide IR transmission has been synthesized. More importantly, compared to the maternal oxide, Sr₂ZnSi₂O₇, the second harmonic generation (SHG) response of Sr₂ZnSn₂OS₆ is enhanced by two orders of magnitude. In addition, Sr₂ZnSn₂OS₆ can exhibit a large band-gap and high laser damage threshold. These advantages make Sr₂ZnSn₂OS₆ a promising IR NLO crystal. Our research will provide insights into the rational design of new IR NLO crystals.

Based on heteroanionic structure engineering, the first heteroanionic thiostannate has been successfully synthesized. It is a promising IR-NLO material exhibiting moderate second-harmonic generation and wide IR transmission.     

Rational design of light-controllable polymer micelles

Amphiphilic block copolymer (BCP) micelles are nanocarriers that hold promise for controlled delivery applications. This account highlights our recent works on light-dissociable BCP micelles. We have designed and developed light-responsive amphiphilic BCPs whose micellar aggregates (core-shell micelles and vesicles) can be disrupted by light exposure. The basic strategy is to incorporate a chromophore into the structure of the hydrophobic block, whose photoreaction can result in a conformational or structural change that shifts the hydrophilic/hydrophobic balance toward the destabilization of the micelles. Using various chromophores including azobenzene, pyrene and nitrobenzene, we have achieved both reversible and irreversible dissociation of BCP micelles upon illumination with UV/visible or near infrared light. The demonstrated rational design principle based on light-changeable or light-switchable amphiphilicity is general and can be applied to many polymer/chromophore combinations. This opens the door to developing photocontrollable polymer nanocarriers offering control over when and where the release of loaded agents takes place.     

Simulation of carbohydrate-protein interactions: Computer-aided design of a second generation GM1 mimic

The oligosaccharide of ganglioside GM1 [Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer] is the cellular target of two bacterial enterotoxins: the cholera toxin (CT) and the heat-labile toxin of E.coli (LT). We recently reported that the pseudosaccharide 2[Gal1-3GalNAc1-4(NeuAc2-3)DCCHD] is a high-affinity ligand for CT, and thus a functional mimic of GM1 (Bernardi, A., Checchia, A., Brocca, P., Sonnino, S. and Zuccotto, F., J. Am. Chem. Soc., 121 (1999) 2032–2036). In this paper we describe the design of a second-generation mimic, formally obtained from 2 by inverting the configuration of a single stereocenter, thus transforming a N-acetyl galactosamine into a N-acetyl glucosamine. The design process involved modeling of the free ligand and its LT complex, followed by qualitative and quantitative comparison with the corresponding structures of 2. The protocol employed relied on both conformational search and molecular dynamics methodologies to account for the flexibility of both the ligand and the protein receptor. The conformational search of the LT:inhibitor complex showed that, compared to 2, the new compound can insert one more hydroxy group within the protein binding site. Molecular dynamics simulations showed that, in turn, this may trigger a series of rearrangements and reorientations of side chains and crystallographic water molecules in the toxin, leading to new H-bond contacts which may result in enhanced affinity of the new inhibitor. FEP calculations were performed by mutating the structure of 2 in solution and in the protein complex, and the prediction was made that the second-generation mimic should be a stronger binder than its parent compound.